Extraction of a substance from dry citrus meal which increases beta-carotene production by blakeslea trispora



United States Patent Ofiice 3,079,38 Patented Feb. 26, 1963 EXTRACTZQNfil A SUESTANQE FRGM BRY \CTT- RUS MEAL WERE INQREAEES fl-QARGTENEPRODUCTlitl-N iii! BL ZKESLEA TRl'SPcQRA Alex (Ziegler, George E. N.Nelson, and Harlow H. Hall, Peoria, TEL, assignors to the United Statesof America as represented by the Secretary of Agriculture No Drawing.Qriginal application Jan. 25, M62, Ser. No. 163,849. Divided and thisapplication May 21, 1952, Ser. No. 265,143

1 Claim. (Cl. 26t)--236.5)

(Granted under Title 35, US. lode (1952), sec. 2.66)

This application is a division of copending application Serial No.168,849, filed January 25, 1962.

A nonexclusive, irrevocable, royalty-free license in the inventionherein described, throughout the world for all purposes of the UnitedStates Government, with the power to grant sublicenses for suchpurposes, is hereby granted to the Government of the United States ofAmerica.

This invention relates to a method of greatly improving the productionof beta-carotene by fermenting a culture of Blakesiea trispora in thepresence of a hitherto unsuspected water-soluble principle extractedfrom citrus waste material.

More particularly this invention relates to the unobvious discovery thatbeta-ionone, the extremely high price of which presently accounts forover half of the total cost of microbiologically producingbeta-carotene, may be substituted by an aqueous extract of extremelylowcost citrus peel waste material. The chemical nature of thewater-soluble material is not known but chromatographic studies and itsinsolubility in organic solvents preclude its being preformedbeta-carotene or a carotenoid-type compound.

The principal object of our invention is the discovery of an inexpensivematerial that can be substituted for beta-ionone in the microbiologicalproduction of betacarotene.

Another object is the discovery of an improved fermentation medium forthe production of beta-carotene.

Yet another object is a process for greatly increasing the yields ofbeta-carotene at a much lower cost than is ,resently known.

Gther objects and advantages will become apparent in the followingdisclosure and appended claim.

In accordance with the obiects of our invention we have now disc veredthat a beta-carotene promoting carotenoid-free aqueous extract ofcommercially available ci rus meal (citrus peel waste that has beentreated with alkali) can be prepared by extracting the dry commercialmeal with certain organic solvents to remove all oilsoluble constituentsincluding any carotenoids or carotenoid-like materials and thenhomogenizing the organic solvent-extracted meal in hot Water to providean aqueous extract. The crude solvent-extracted homogenate may be addedto the fermentation medium as is, or the solids may be removed therefromin any convenient manner and concentrated or water conveniently added tomake 1 ml. represent 1 gm. of citrus meal.

In addition commercially available citrus molasses (obtained from wholecitrus peel by treating the peel with alkali and then pressing out theaqueous molasses from the peel) and which is free of inherentcarotenoids or carotenoid-iike materials, may be substituted for thebcta-ionone to obtain comparably increased yields of beta-carotene.

Solvent-extracted citrus peel aqueous homogenates (orange, tangerine,lemon, lime, grapefruit, or mixtures thereof) can be substituted for thebeta-ionone with essentially similar results, but the unextracted wholepeel or aqueous homogenate thereof is much less active because it alsoapparently contains an oil-soluble component that inhibits thefermentation.

We also determined that the addition of the fermentation broth ofpro-formed beta-carotene did not improve the yield, said additionapparently not being metabolized so as to be protected from destructionby incorporation into the mycelia. Also, the addition of otherwise nontoxic solvents containing only the traces of carotene and xanthophylspresent in the whole peel were apparently toxic to the organism inasmuch.as beta-carotene production was greatly reduced.

The following examples and data typify the remarkably improved resultsof our invention.

EXAMPLE 1 100 g. of commercially obtained dry citrus meal was extractedwith 600 ml. acetone in a Soxhlet apparatus until there was no furtherremoval of pigment (about 34 hours). The acetone was discarded and thecitrus solids in the extractor thirnble were similarly extracted with600 ml. of an equal mixture of pentane and hexane (B.P. 33-57 C.), andspread out to air dry. About 700-800 ml. of distilled water was thenadded to the solvent-extracted solids, the aqueous dispersion then beingboiled for about 30 minutes. The solids were separated by filtration andrepeatedly washed with 100 ml. aliquots of boiling water until thelatter became quite clear. All of the aqueous portions were combined andthen conentrated to 100 ml. by flash evaporation at 35 C., the resultingconcentrate then being added to the fermentation in place ofbeta-ionone. Respectively 5 parts and 10 parts of our aqueous extractwere added in respective 500 ml. conical flasks to 100 parts (100 ml.)of a fer mentation medium having the following composition per 1000 ml:

Tap water, q.s. ad 1000 ml.

The flasks then were plugged, sterilized, and cooled and each was theninoculated with 4 ml. of each of a 48 hr. stock culture of Blakcsleatrispora NRRL 9216 and Blakeslea trispora NRRL 9159. The flasks werethen agitated at 28 C. on a Gump shaker at 200 r.p.m. for 144 hours, themycelia from each flask then being harvested by filtration, dried in avacuum oven at (3., ground to a powder in a Wiley mill, dissolved inpetroleum ether (B.P. 3357 C.), and the solutions comparedspectrophotometrically with an authentic beta-carotene sample in thesame solvent. The yields compared with that of a control containing nocitrus peel extract are shown in Table I.

EXAMPLE 2 The fermentation of Example 1 was repeated with the exceptionthat commercial citrus molasses was substituted forthe aqueous extractof citrus meal. The results are Having disclosed our invention, weclaim: shown in Table 11. Method of preparing an extract that increasesthe pro- T able II duction of beta-carotene bytBlakeslea trisporacomprising the steps of successively extracting commercial dry Gm, DryBeta- 5 citrus meal with acetone and then with an equal mixture Citrus111013555 d t fi myccliad 7 of pcntane and hexane, removing the organicsolvents, extracting the residue with successive portions of boilingWater, pooling the aqueous portions, and concentrating the pooledaqueous extracts so that 1 ml. corresponds to S g t $38 10 1 gm. of thedry citrus meal.

5-.0- v 101,100 No references cited.

